The present invention relates generally to a unique vertebrate protein, tankyrase that binds to telomeric repeat binding factor 1 (TRF1), to the nucleic acids encoding tankyrases, and to therapeutic methods of use thereof. The tankyrases may also have a particular use in developing drugs that can counteract the telomere shortening associated with aging and certain diseases such as ataxia telangiectasia.
Telomeres are terminal structural elements found at the end of chromosomes [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939)] that protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock, Genetics, 26:234-282 (1941); Lundblad et al., Cell, 87:369-375 (1996)]. Telomeres are also thought to play a role in the architecture of the nucleus [Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J., 10:214-330 (1885)], and to provide a solution to the end-replication problem that arises as a consequence of successive replication of linear DNA by DNA polymerases which would otherwise result with progressively shorter terminal sequences [Watson, Nature, 239:197-201 (1972)]. In tetrahymena, impaired telomere function leads to a defect in cytokinesis and to cell death [Yu et al., Nature, 344:126-132 (1990)]. Similarly, in yeast, loss of a single telomere results in cell cycle arrest and chromosome instability [Sandell and Zakian, Cell, 75:729-741 (1993)] and cells undergoing generalized telomere shortening eventually senesce [Lundblad and Szostak, Cell, 57:633-643 (1989); Singer and Gottschling, Science, 266:404-409 (1994)].
A ribonucleoprotein reverse transcriptase, telomerase, can elongate telomeres using an internal RNA component as template for the addition of the appropriate G-rich sequence to the 3xe2x80x2 telomere termini [Greider and Blackburn, Cell, 43:405-413 (1985)]. This activity is thought to compensate for the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al., Nature, 337:429-433 (1989)].
Telomeres contain a tandem array of repeat sequences, typically five to eight base pairs long, that are G-rich in the strand that extends to the end of the chromosome DNA. These repeat units appear to be both necessary and sufficient for telomere function [Lundblad and Szostak, Cell, 57:633-643 (1989); Szostak et al., Cell, 36:459-568 (1982)]. All telomeres of a single genome are composed of the same repeats and these sequences are highly conserved across species. For instance, Oxytricha chromosomes terminate in TTTTGGGG repeats [Klobutcher et al., Proc. Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG)n [Blackburn et al., J. Mol. Biol., 120:33-53 (1978)], and plant chromosomes carry the sequence (TTTAGGG)n [Richards et al., Cell, 53:127-136 (1988)]. Telomeres of trypanosomes and all vertebrates, including mammals, contain the repeat sequence TTAGGG [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature, 338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis et al., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg et al., Cell, 36:459-468 (1984)]. This 6 basepair sequence is repeated in long tandem arrays at the chromosome ends, which may be as long as 100 kb in the mouse, and varies from 2 to 30 kb in humans [de Lange, Telomere Dynamics and Genome Instability in Human Cancer, In Telomeres, Blackburn and Greider eds., Cold Spring Harbor Press; 265-295 (1995)].
During the development of human somatic tissue, telomeres undergo progressive shortening; in contrast, sperm telomeres increase with donor age [Broccoli et al., Proc. Natl. Acad. Sci. USA, 92:9082-9086 (1995); de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Most if not all human somatic tissue chromosomes lose terminal TTAGGG repeats with each division, e.g., about 15-40 basepairs per year in the skin and blood. It is unclear what effect this diminution has since human telomeres are between 6-10 kb at birth. On the other hand, it is not yet known how many kilobases of TTAGGG repeats are necessary for optimal telomere function.
Primary human fibroblasts grown in culture lose about 50 basepairs of telomeric DNA per doubling (PD) before they stop dividing at a senescence stage [Allsopp et al., Proc. Natl. Acad. Sci. USA, 89:10114-10118 (1992)]. Importantly, there is an excellent correlation between the number of divisions that the cells go through and their initial telomere length. Indeed, it has been suggested that the correlation represents a molecular clock, which limits the potential of primary cells to replicate [Harley et al., Nature (London), 345:458-460 (1990); Harley et al., Exp. Gerontol, 27:375-382 (1992)]. Thus, immortalization of human somatic cells involves a mechanism to halt telomere shortening [Bodnar et al., Science, 279:349-352 (1998)].
Changes in telomeric dynamics also appear to play a role in the malignant transformation of human cells [Counter et al., EMBO J., 11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci. USA, 91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994)]. For example, telomeres of tumor cells are generally significantly shorter than those of the corresponding normal cells [de Lange et al., Mol. Cell Biol., 10:518-527 (1990)]. Telomerase activation appears to be an obligatory step in the immortalization of human cells [de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994); Counter et al., EMBO J., 11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci., 91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994); Bodnar et al., Science, 279:349-352 (1998)].
Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the sequence dependence of telomere formation matched the in vitro binding requirements for TRF1, a telomeric TTAGGG repeat binding protein that is associated with human and mouse telomeres in interphase and in mitosis.
Indeed, several observations suggest the existence of regulatory mechanisms to control telomere length. Mammalian telomeres show a species-specific length setting [Kipling and Cooke, Nature, 347:400-402 (1990)] indicating a mechanism to control telomere length in the germline. Mammalian cells also have a mechanism to measure and regulate the length of individual telomeres. For example, in telomere seed experiments the final length of individual newly-formed telomeres matches the length of the host cell telomeres [Barnett et al., Nucl. Acids Res., 21:27-36 (1993); Hanish et al., Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)]. Telomere length regulation is also apparent in several human cell lines, which maintain their telomeres at a stable length setting despite high levels of telomerase [Counter et al., EMBO J., 11: 1921-1929 (1992)]. Thus, cells can monitor and modulate individual telomeres, a process that is likely to involve proteins bound to the TTAGGG repeats at chromosome ends.
Another process likely to be mediated by TTAGGG binding proteins is the protective cap function of telomeres. Telomeres are protected from the cellular surveillance systems that monitor DNA damage. Thus, cells can distinguish natural chromosome ends (telomeres) from double strand breaks (resulting from DNA damage).
The only known protein components of mammalian telomeres are the TRF proteins, duplex TTAGGG repeat binding factors that are localized at telomeres in interphase and metaphase chromosomes [Zhong et al., Mol. Cell. Biol., 13:4834-4943 (1992); Chong et al., Science, 270:1663-1667 (1995); Ludxc3xa9rus et al., J. Cell Biol., 135:867-881 (1996); Broccoli et al., Hum. Mol. Genetics, 6:69-76 (1997); see Smith and de Lange, Trends in Genetics, 13:21 -26 (1997) for review; Broccoli et al., Nature Gen., 17:231-235 (1997); Bilaud et al., Nature Gen., 17:236-239 (1997); van Steensel et al., Cell, 92:401-413 (1998)]. Thus far, only two human telomeric DNA binding proteins have been identified, TRF1 and TRF2 [U.S. Pat. No. 5,733,730, Issued Mar. 31, 1998, and U.S. patent application Ser. No: 08/938,052, filed Sep. 26, 1997, and Ser. No. 09/018,636 filed Feb. 4, 1998, all of which are whereby incorporated by reference in their entireties]. TRF1 was isolated as a double-stranded TTAGGG-repeat binding protein from HeLa cells [Chong et al., Science, 270:1663-1667 (1995)]. This factor contains three recognizable domains: an acidic N-terminal domain, a dimerization domain, and a C-terminal three helix bundle similar to the Myb and homeodomain DNA-binding folds [Bianchi et al., EMBO J., 16:1785-1794 (1997); Chong et al., Science, 270:1663-1667 (1995); reviewed in Konig and Rhodes, Cell, 85:125-136 (1996); Smith and de Lange, Trends in Genetics, 13:21-26 (1997)]. A second factor, TRF2, is related to TRF1 in its dimerization domain and the C-terminal Myb motif, but differs in that its N-terminus is basic rather than acidic [Bilaud et al., Nature Gen., 17:236-239 (1997); Broccoli et al., Nature Gen., 17:231-235 (1997)]. Despite their related dimerization domains, the proteins do not interact with each other [Broccoli, et al., Nature Gen., 17:231-235 (1997)], and probably exist predominantly as homodimers. Both proteins bind specifically to double-stranded TTAGGG repeats in vitro and are located at telomeres in vivo. The two TRFs are ubiquitously expressed and current evidence indicates that most human telomeres contain both factors bound simultaneously throughout the cell cycle [Broccoli et al., Nature Gen., 17:231-235 (1997); Chong et al., Science, 270:1663-1667 (1995); Smith and de Lange, Trends in Genetics, 13:21-26 (1997)]. Two other double-stranded telomeric-repeat binding proteins have been identified; Rap1p in S. cerevisia [Reviewed in Shore, Trends Gen., 10:408-412 (1994) and Tazlp in S. pombe [Cooper et al., Nature, 385:744-474 (1997)]. Both have Myb type DNA-binding domains [Cooper et al., Nature, 385:744-747 (1997); Konig et al., Cell, 85:125-136 (1996)]. In addition, Tazlp shows weak overall homology with TFR1 and shares its acidic nature [Cooper et al., Nature, 385:744-747 (1997)].
Recent studies have shown that TRF2 plays a key role in the protective activity of telomeres by inhibiting end-to-end fusions [van Steensel et al., Cell, 92:401-413 (1998)]. Previous studies had indicated that TRF1 plays a different role in telomere biology, functioning as a negative regulator of telomere length maintenance [van Steensel and de Lange, Nature, 385:740-743 (1997)]. Thus, long-term overexpression of TRF1 in a telomerase-positive tumor cell line resulted in progressive telomere shortening. Conversely, removal of TRF1 from the telomere (through expression of a dominant negative mutant) induced telomere elongation. In these experiments TRF1 did not detectably alter the activity of telomerase in cell extracts. Based on these observations it was proposed that TRF1 negatively regulates telomerase at the level of individual telomeres; an increase in the amount of TRF1 at the telomere would create a negative signal for telomerase, whereas, a decrease would send a positive signal to telomerase [van Steensel and de Lange, Nature, 385:740-743 (1997)]. Interestingly, a similar mechanism of telomere length regulation exists in yeasts where it has been shown that Taz1p and Rap1p function as negative regulators of telomere length. As is the case for yeast telomere length regulation, the mechanism by which TRF1 controls telomere synthesis by telomerase is not fully understood [Conrad et al., Cell, 63:739-750 (1990); Cooper et al., Nature, 385:744-747 (1997); Lustig et al., Science, 250:549-553 (1990); Marcand et al., Science, 275:986-990 (1997); McEachern and Blackburn, Nature, 376:403-409 (1995)].
Indeed, telomere homeostasis involves a balance of lengthening and shortening activities. The telomerase catalytic subunit produces the lengthening activity, whereas other proteins including the telomere binding protein TRF1 are involved in establishing a telomere length equilibrium. Recently Bodnar et al. [Science 279:349-352 (1998)] have shown that extremely low levels of telomerase activity are insufficient to prevent telomere shortening; a result that is consistent with the observation that stem cells have low but detectable telomerase activity, yet continue to exhibit shortening of their telomeres throughout life.
Therefore, there is a need to isolate additional proteins, preferably enzymes involved in telomere homeostasis. Furthermore, there is a need to characterize such proteins. In addition, there is a need to design and develop drug screens to identify agents that modulate such proteins and thus can act as effectors on the important process of telomere length homeostasis.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides an isolated and/or recombinant nucleic acid encoding a protein, tankyrase, that binds to TRF1. In another embodiment, the nucleic acid encodes a tankyrase-related protein. In one embodiment the nucleic acid encodes a tankyrase or a tankyrase-related protein that has an amino acid sequence that has at least 25% identity with that of SEQ ID NO:2. In another embodiment the nucleic acid encodes a tankyrase or tankyrase-related protein comprising at least two, preferably three and more preferably all of the following domains: a domain that consists of homopolymeric tracts of histidine, proline and serine (HPS) preferably at the amino terminal end of the protein, an ankyrin-specific (ANK) repeat consensus domain, a sterile alpha motif (SAM) motif, and a poly(ADP-ribose) polymerase (PARP)-related domain. Preferably the order of the domains is identical to that found in human tankyrase having the amino acid sequence of SEQ ID NO:2. The tankyrase is preferably an animal protein, more preferably a vertebrate protein, and even more preferably a mammalian protein. In the most preferred embodiment the tankyrase is a human protein. In one such embodiment the protein is about 142-kDaltons and contains 24 ANK repeats, a SAM motif, a PARP-related domain, and an N-terminal domain rich in proline, histidine and serine (HPS). In another such embodiment tankyrase is a protein that is relatively enriched in the nuclear envelope fraction and in a tight association with the nuclear envelope e.g., remaining bound to the nuclear envelope even after extraction with 0.5 M NaCl and 8 M urea. In a particular embodiment of this type the nucleic acid encodes a tankyrase that is a human protein comprising the amino acid sequence of SEQ ID NO:2. In a related embodiment of this type the nucleic acid encodes a tankyrase comprising the amino acid sequence of SEQ ID NO:2 with a conservative amino acid substitution. In a more particular embodiment the nucleic acid comprises the coding sequence of SEQ ID NO:1. All of the recombinant and/or isolated nucleic acids of the present invention can further comprise a heterologous nucleotide sequence.
The present invention also provides nucleic acids, e.g., recombinant DNA molecules that comprise a nucleotide sequence encoding a fragment of a tankyrase that can bind to the acidic domain of a TRF1. In a preferred embodiment the fragment comprises at least a portion of the ANK repeat consensus domain of the tankyrase. In a particular embodiment of this type the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 436 to 796 of SEQ ID NO:2. In a related embodiment of this type the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 436 to 796 of SEQ ID NO:2 with a conservative amino acid substitution. In another such embodiment the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 181 to 1005 of SEQ ID NO:2. In a related embodiment of this type the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 181 to 1005 of SEQ ID NO:2 with a conservative amino acid substitution. In still another embodiment of this type, the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 336 to 1163 of SEQ ID NO:2. In a related embodiment of this type the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 336 to 1163 of SEQ ID NO:2 with a conservative amino acid substitution.
In another embodiment a nucleic acid, e.g., a recombinant DNA molecule comprises a nucleotide sequence encoding a fragment of a tankyrase comprising a PARP-related domain. In one such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1159 to 1314 of SEQ ID NO:2. In another such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1159 to 1314 of SEQ ID NO:2 with a conservative amino acid substitution.
In still another embodiment a nucleic acid e.g., a recombinant DNA molecule, comprises a nucleotide sequence encoding a fragment of a tankyrase comprising a SAM motif. In one such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1023 to 1088 of SEQ ID NO:2. In another such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1023 to 1088 of SEQ ID NO:2 with a conservative amino acid substitution. As is true for all of the nucleic acids of the present invention, all of the recombinant DNA molecules encoding fragments of a tankyrase can further comprise a heterologous nucleotide sequence.
In yet another embodiment, a nucleic acid, e.g., a recombinant DNA molecule comprises a nucleotide sequence encoding a fragment of tankyrase comprising an HPS domain. In one such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1-180 of SEQ ID NO:2. In another such embodiment the nucleic acid comprises a nucleotide sequence encoding a fragment of a tankyrase comprising the amino acids 1-180 of SEQ ID NO:2 with a conservative amino acid substitution.
The present invention also provides nucleic acids, e.g., recombinant DNA molecules that comprise a nucleotide sequence encoding a truncated tankyrase. In one such embodiment the nucleotide sequence comprises the coding sequence for amino acid residues 1-640 of SEQ ID NO:2. In another embodiment, the nucleotide sequence comprises the coding sequence for amino acid residues 1-881 of SEQ ID NO:2. In one embodiment the nucleotide sequence encodes SEQ ID NO:8 or SEQ ID NO:8 with a conservative amino acid substitution. In a particular embodiment of this type the nucleic acid has the nucleotide sequence of SEQ ID NO:7. In another embodiment the nucleotide sequence encodes SEQ ID NO:10 or SEQ ID NO:10 with a conservative amino acid substitution. In a particular embodiment of this type the nucleic acid has the nucleotide sequence of SEQ ID NO:9.
Nucleic acids that hybridize to the nucleotide sequences that encode the tankyrases, fragments thereof including truncated tankyrases, tankyrase-related proteins, and fragments thereof are also included in the present invention. In one such embodiment the nucleic acid is at least about 24 nucleotides, preferably at least about 48 nucleotides, and more preferably at least about 96 nucleotides. In a preferred embodiment of this type, the nucleic acid encodes a tankyrase which has at least one functional activity, preferably two and more preferably all, of the activities of human tankyrase as disclosed herein. In a particular embodiment the nucleic acid hybridizes to SEQ ID NO:1 under moderately stringent conditions. In a preferred embodiment of this type, the nucleic acid hybridizes to SEQ ID NO:1 under high stringency conditions.
The present invention further provides a nucleic acid that comprises about 15 or more, preferably about 24 or more, and more preferably about 36 or more consecutive nucleotides from SEQ ID NO:1. In a preferred embodiment of this type, the nucleic acid encodes a tankyrase which has at least one functional activity, preferably two, and more preferably all of the functional activities of human tankyrase as disclosed herein.
In addition, the present invention also provides nucleotide probes for the isolated and/or recombinant nucleic acids of the present invention. In a preferred embodiment of this type the nucleotide probe is for SEQ ID NO:1. Another nucleic acid that can be used as a probe contains the nucleotide sequence of SEQ ID NO:11. Still another nucleic acid that can be used as a probe contains the nucleotide sequence of SEQ ID NO:12.
All of the nucleic acids of the present invention can be comprised by a recombinant DNA molecule that is operatively linked to an expression control sequence. The present invention further provides expression vectors containing the recombinant DNA molecules of the present invention. In addition the present invention also provides methods of expressing a recombinant tankyrase protein or fragment thereof in a cell containing an expression vector of present invention. One such embodiment comprises culturing the cell in an appropriate cell culture medium under conditions that provide for expression of recombinant tankyrase or fragment thereof by the cell. Such methods can further include the step of purifying the recombinant tankyrase or fragment thereof. The purified form of the recombinant tankyrases or fragments thereof are also included as part of the present invention. In one preferred embodiment the nucleic acid encodes SEQ ID NO:2. In another preferred embodiment, the nucleic acid encodes a fragment of the tankyrase that comprises the amino acids 436 to 796 of SEQ ID NO:2.
Another aspect of the present invention provides an isolated and/or recombinant protein, tankyrase, that binds to TRF1. In another embodiment, the isolated and/or recombinant protein is a tankyrase-related protein. In one embodiment the tankyrase or tankyrase-related protein has an amino acid sequence that has at least 25% identity with that of SEQ ID NO:2. In another embodiment the tankyrase or tankyrase-related protein comprises at least two, preferably three, and more preferably all of the following domains: a domain rich in homopolymeric tracts of histidine, proline, and serine (HPS) which is preferably at the amino-terminal end of the protein, an ankyrin-specific (ANK) repeat consensus domain, a sterile alpha motif (SAM) motif, and a poly(ADP-ribose) polymerase (PARP)-related domain. The tankyrase is preferably an animal protein, more preferably a vertebrate protein, and even more preferably a mammalian protein. In the most preferred embodiment the tankyrase is a human protein. In one such embodiment the protein is about 142-kDaltons and contains about 24 ANK repeats, a SAM motif, an amino-terminus rich in histidine, proline and serine (i.e., an HPS domain), and a PARP-related domain. In another such embodiment the tankyrase is a protein that is relatively enriched in the nuclear envelope fraction and in a tight association with the nuclear envelope e.g., remaining bound to the nuclear envelope even after extraction with 0.5 M NaCl and 8 M urea.
In another embodiment the present invention provides a tankyrase that is a human protein comprising the amino acid sequence of SEQ ID NO:2. In a related embodiment of this type the tankyrase comprises the amino acid sequence of SEQ ID NO:2 with a conservative amino acid substitution. The present invention further provides proteolytic fragments of the tankyrase proteins of the present invention. The present invention also provides a protein comprising about 12 or more, preferably about 24 or more, and more preferably about 36 or more consecutive amino acids from SEQ ID NO:2 which functions as a tankyrase as disclosed herein.
The present invention also provides a fragment of a tankyrase that can bind to the acidic domain of a TRF1. In a preferred embodiment the fragment comprises at least a portion of the ANK repeat consensus domain of the tankyrase. In a particular embodiment of this type the fragment of the tankyrase comprises the amino acids 436 to 796 of SEQ ID NO:2. In a related embodiment of this type the fragment of the tankyrase comprises the amino acids 436 to 796 of SEQ ID NO:2 with a conservative amino acid substitution. In another such embodiment the fragment of the tankyrase comprises the amino acids 181 to 1005 of SEQ ID NO:2. In a related embodiment of this type the fragment of the tankyrase comprises the amino acids 181 to 1005 of SEQ ID NO:2 with a conservative amino acid substitution. In still another embodiment of this type, the fragment of the tankyrase comprises the amino acids 336 to 1163 of SEQ ID NO:2. In a related embodiment of this type the fragment of the tankyrase comprises the amino acids 336 to 1163 of SEQ ID NO:2 with a conservative amino acid substitution.
In still another embodiment a fragment of the tankyrase comprises an HPS domain. In one such embodiment the fragment of a tankyrase comprises the amino acids 1 to 180 of SEQ ID NO:2. In another such embodiment the fragment of a tankyrase comprises the amino acids 1 to 180 of SEQ ID NO:2 with a conservative amino acid substitution.
In yet another embodiment a fragment of a tankyrase comprises a PARP-related domain. In one such embodiment the fragment of a tankyrase comprises the amino acids 1159 to 1314 of SEQ ID NO:2. In another such embodiment the fragment of a tankyrase comprises the amino acids 1159 to 1314 of SEQ ID NO:2 with a conservative amino acid substitution.
In still another embodiment a fragment of a tankyrase comprises a SAM motif. In one such embodiment the fragment of a tankyrase comprises the amino acids 1023 to 1088 of SEQ ID NO:2. In another such embodiment the fragment of a tankyrase comprises the amino acids 1023 to 1088 of SEQ ID NO:2 with a conservative amino acid substitution. All of the recombinant and/or isolated tankyrase proteins and fragments of the present invention can further be part of a chimeric and/or fusion peptide or protein.
The present invention also provides truncated tankyrases. In one such embodiment the truncated tankyrase comprises amino acid residues 1-640 of SEQ ID NO:2. In another embodiment, the truncated tankyrase comprises amino acid residues 1-881 of SEQ ID NO:2. In one embodiment the truncated tankyrase comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 with a conservative amino acid substitution. In yet another embodiment the truncated tankyrase comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:10 with a conservative amino acid substitution.
The present invention further provides antibodies to the proteins and fragments thereof including truncated proteins, and proteolytic fragments of the proteins of the present invention. In one such embodiment the antibody is a polyclonal antibody. In another embodiment the antibody is a monoclonal antibody. In still another embodiment the antibody is a chimeric antibody. The present invention further provides an immortal cell line that produces a monoclonal antibody of the present invention.
In another aspect of the present invention is a method of selecting a candidate drug that interferes with the binding of a tankyrase and a TRF1. One such embodiment comprises contacting a candidate drug with a first protein or peptide comprising the acidic domain of a TRF1 and a second protein or peptide comprising a tankyrase fragment that can bind to the acidic domain of a TRF1 under conditions where the first protein or peptide and second protein or peptide bind in the absence of the candidate drug and determining the binding between the first protein or peptide and second protein or peptide; wherein a candidate drug is selected when the amount of binding determined in the presence of the drug is measurably less than in its absence. Preferably the fragment comprises at least a portion of the ANK repeat consensus domain of the tankyrase.
The present invention further provides methods of selecting a candidate drug that can modulate the PARP (and/or ARP) activity of a tankyrase. Such modulators can be either agonists or antagonists. Candidate drugs that are selected as agonists cause an increase in PARP (or ARP) activity whereas candidate drugs that are selected as antagonists (e.g., inhibitors) cause a decrease in PARP (and/or ARP) activity. One such embodiment comprises contacting a candidate drug with a tankyrase or a fragment of tankyrase that has PARP activity, NAD+ and a poly ADP-ribosylating substrate under conditions in which the tankyrase (or the fragment) polyADP-ribosylates the substrate in the absence of the candidate drug. The polyADP-ribosylation state of the substrate (e.g., a histone) is then determined. A candidate drug is selected as an antagonist when the polyADP-ribosylation state of the substrate determined in the presence of the drug is measurably less than its absence. A candidate drug is selected as an agonist when the polyADP-ribosylation state of the substrate determined in the presence of the drug is measurably greater than its absence.
The present invention further provides methods of extending the lifespan of a non-tumor cell and/or inhibiting the growth of a tumor cell. One such embodiment comprises administering an inhibitor to tankyrase. In one particular embodiment of this type the inhibitor is 3-aminobenzamide. In a preferred embodiment of this type the cell is a human cell.
Yet another aspect of the present invention comprises a method of identifying the sequence of a homologue to the human tankyrase gene. One such embodiment comprises determining the homology of SEQ ID NO:2 to the amino acid sequences encoded by nucleic acids from a library of nucleic acids containing partial nucleotide sequences of coding regions of genes. Preferably this determination is aided by computer analysis. A nucleic acid containing a partial nucleotide sequence encoding a protein that is substantially homologous to SEQ ID NO:2 is then selected. The sequence of the coding region of the gene is then determined. The sequence is identified as being that of the homologue to the human tankyrase gene of the invention when it encodes a protein having an amino acid sequence that is substantially homologous to SEQ ID NO:2.
In one embodiment of the method, determining the sequence of the coding region is performed by sequencing an insert of a plasmid which contains the nucleic acid. In this case, the insert comprises the nucleic acid. In another embodiment, the method further comprises constructing a recombinant DNA that contains the coding region. In one such embodiment a recombinant protein is made by expressing the recombinant DNA. In a preferred embodiment of this type an activity of the tankyrase is assayed. In one such embodiment, the activity assayed is the ability of the recombinant protein to bind to TRF1. In another embodiment the sequence is identified as being that of the homologue to the human tankyrase gene when the recombinant protein has the activity of the human tankyrase. Recombinant DNA molecules and the recombinant tankyrases obtained by these methods are also part of the present invention.
The present invention further provides a method of transporting a protein to the nucleus. This method arises from the identification of the mechanism by which tankyrase is carried into the nucleus by TRF1. More particularly, the present invention provides a nucleotide sequence that encodes a protein or peptide of interest and a tankyrase fragment that can bind to the acidic domain of a TRF1. Minimally the fragment of tankyrase comprises at least a portion of the ANK repeat consensus domain. A particularly useful aspect of this portion of the present invention is that the protein of interest can be localized to the telomere. Such a protein can be used as a marker such as green fluorescent protein, or for its particular activity such as a particular RNase, Dnase, or even a protein kinase. In a preferred embodiment of this type the nucleic acid encodes a fragment of the tankyrase comprising the amino acids 436 to 796 of SEQ ID NO:2. In another embodiment of this type the nucleic acid encodes a fragment of the tankyrase comprising the amino acids 436 to 796 of SEQ ID NO:2 with a conservative amino acid substitution.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.